Transplatinum complexes with N2O2 donor sets as cytotoxic and antitumor agents

ABSTRACT

Trans-platinum compounds comprising carboxylato groups are disclosed, with anti-cancer usefulness. The carboxylato groups participate in solubilizing trans-platinum compounds to which they are attached.

RELATED APPLICATION

This application claim priority from U.S. provisional application Ser.No. 60/707,176 filed Aug. 11, 2005 for “Trans-platinum complexes withN₂O₂ donor sets as cytotoxic and antitumor agents” by Nicholas Farrell.

STATEMENT REGARDING FUNDING

This invention was made using funds from grants from the NationalScience Foundation having grant number CHE034205. The United Statesgovernment may have certain rights in this invention.

FIELD OF THE INVENTION

The invention generally relates to novel cytotoxic and antitumorplatinum II compounds. In particular, the invention providestrans-platinum complexes with N₂O₂ donor sets which are stable, watersoluble, and display a low level of cross resistance with traditionalcis-Pt anticancer drugs.

BACKGROUND OF THE INVENTION

Current anti-cancer drugs based on platinum (cisplatin (i.e.,cis-[PtCl₂(NH₃)₂], cis-DDP); carboplatin (i.e., cis-[Pt(CBDCA)(NH₃)₂],(CBDCA=cyclobutane-1,1-dicarboxylate)); and oxaliplatin (i.e.,[Pt(ox)(dach)] (dach=1,2-diaminocyclohexane, ox=oxalato)) share similarclinical properties due to their structural similarities (namely, theircis geometry around the platinum atom). Their mechanisms of action,especially with respect to the purported target DNA, are alsoconsequently very similar. While these are the drugs of choice for thetreatment of many types of tumors, with time, tumor cells developresistance to their cytotoxic effects. The hope in the search for newplatinum-based drugs with novel pharmacological properties lies infinding structurally new compounds with fundamentally differentmechanisms of action. Such drugs would be useful both on a stand-alonebasis for cancer therapy, and would be of particular interest fortreating tumors that have become resistant to traditional Pt-baseddrugs.

For a long time, it was widely believed that, to be cytotoxic andantitumor active, a platinum drug must have a cis geometry with generalformula cis-[PtX₂(am(m)ine)₂] where X is considered the leaving group,usually chloride or X₂ is a bidentate carboxylate such as1,1-cyclobutanedicarboxylate (CBDCA) or oxalate. The am(m)ine is thedonor ligand and is often NH₃ or (amine)₂ can be a bidentate amine suchas 1,2-diaminocyclohexane. The three clinically approved drugs in theUnited States—cisplatin, carboplatin and oxaliplatin—are all covered bythis general formula.

One approach to expand the anticancer spectrum of platinum agents hasbeen to examine structurally unique platinum agents. Through thisapproach, polynuclear platinum compounds (e.g., trinuclear BBR3464) haveemerged as a class. (Farrell, N: Polynuclear Drugs. Metal Ions in Biol.Sys. 41:252-296 (2004); Farrell, N.: Platinum Anti-cancer drugs. FromLaboratory to Clinic. ACS Symposium Series 903 “Medicinal InorganicChemistry,” Sessler, J. A., Doctorow, S. E., McMurry, T. J. and Lippard,S. J. Eds., 62-79 (2005).

Another approach has been to explore the trans geometry. Farrell, N.,Current status of structure-activity relationships of platinumanti-cancer drugs: activation of the trans geometry, Met. Ions Biol.Syst., 1996, 32, 251-296; Intini, F. P.; Boccarelli, A.; Francia, V. C.;Pacifico, C. Sivo, M. F.; Natile, G.; Giordano, D.; Rinaldis, P.;Coluccia, M., Platinum complexes with imino ethers or cyclic ligandsmimicking imino ethers: synthesis, in vitro antitumour activity, and DNAinteraction properties, J. Biol. Inorg. Chem., 2004, 9, 768-780. Theparadigm for the early structure activity relationships (SARs) was thatthe trans geometry, trans-[PtCl₂(NH₃)₂] (trans-DDP) was therapeuticallyinactive.

Substitution of the NH₃ group by a sterically hindered planar aminetrans-[PtCl₂(L)(L′)] (L=NH₃, L′=pyridine, quinoline, thiazole, etc.and/or L=L′=pyridine or thiazole) gives transplanaramine (TPA) compoundswith cytotoxity to cisplatin in human tumor cell-lines. (van Beusichem,M.; Farrell, N., Activation of the trans geometry in platinum antitumorcomplexes. Synthesis, characterization, and biological activity ofcomplexes with the planar ligands pyridine, N-methylimidazole, thiazole,and quinoline. Crystal and molecular structure oftrans-dicholorobis(thiazole)platinum(II), Inorg. Chem. 1992, 31,634-639; Farrell, N.; Kelland, L. R.; Roberts, J. D.; van Beusichem, M.,Activation of the trans geometry in platinum antitumor complexes: asurvey of the cytotoxity of trans complexes containing planar ligands inmurine L1210 and human tumor panels and studies on their mechanism ofaction, Cancer Res., 1992, 52, 5056-5072.) Use of the planar amineenhances cytotoxicity up to 100-fold over trans-[PtCl₂(NH₃)₂]. Further,the compounds generally maintain cytotoxicity in cisplatin-resistantlines.

Since those initial reports by Farrell et al., other groups confirmedthe effects of a sterically demanding group in modulation of thecytotoxicity of the transplatinum structure—amines used includecyclohexylamine (Mellish, K. J.; Barnard, C. F. J.; Murrer, B. A.;Kelland, L. R., DNA-binding properties of novel cis- andtrans-platinum-based anticancer agents in 2 human ovarian carcinoma celllines, Int. J. Cancer, 1995, 62, 717-723), branched aliphatic amines(Monterro, E. I.; Diaz, S.; Gonzalez-Vadillo, A. M.; Perez, J. M.;Alonso, C., Navarro-Ranninger, C., Preparation and Characterization ofNovel trans-[PtCl₂(amine)(isopropylamine)] Compounds: Cytotoxic Activityand Apoptosis Induction in ras-Transformed Cells, J. Med. Chem., 1999,42, 4264-4268), piperzine, piperidine (Khazanov E.; Barenholz Y.; GibsonD.; Najajreh Y., Novel apoptosis-inducing trans-platinum piperidinederivatives: synthesis and biological characterization, J. Med. Chem.2002, 45, 5196-204; Najajreh Y.; Perez J. M.; Navarro-Ranninger, C.;Gibson, D. Novel soluble cationic trans-diaminedichloroplatinum(II)complexes that are active against cisplatin resistant ovarian cancercell lines, J. Med. Chem. 2002, 45, 5189-95) and iminoethers (Intini etal., supra). In general the cytotoxicity of these various compounds isin the 1-20 μM range and is characterized by lack of cross-resistance tocisplatin. The DNA binding profiles of these various compounds show awide diversity in comparison to those of the cisplatin family. Thediscovery that trans compounds of formula trans-[PtX₂(amine)₂] ortrans-[PtX₂ (iminoether)₂] can also be cytotoxic has opened a newopportunity in the search of new drugs with cytotoxicity profilescomplementary to that of the known clinical agents. Substitution of theNH₃ group by a sterically hindered planar amine trans-[PtCl₂(L)(L′)](L=NH₃, L′=pyridine, quinoline, thiazole, etc. and/or L=L′=pyridine orthiazole) gives “transplanaramine” (TPA) compounds with cytotoxicitycomparable to cisplatin in human tumor cell lines. A study of thecytotoxicity of the trans-[PtCl₂(L)(L′)] series across the NCI humantumor cell line panel showed a unique profile and activity in bothcisplatin and oxaliplatin-resistant cells (Fojo T, Farrell, N, Ortuzar,W., Tanimura, H., Weinstein, J., and Myers, T. G.: Identification ofnon-cross-resistant platinum compounds with novel cytoxicity profilesusing the NcI anticancer drug screen and clustered image mapvisualizations. Crit. Revs. Oncol./Hematool. 53:25-34 (2005).) Asstated, all modifications of transplatinum compounds reported to dateare of the form trans-[PtCl₂(L)(L′)] where L and L′ are various aminesother than NH₃. Unfortunately, these compounds are poorly soluble inaqueous medium and thus their in vivo bioavailability is attenuated. Inaddition, these known compounds still contain the relatively reactiveCl—Pt—Cl axis, making them unstable in the cellular environment.(McGowan, G.; Parsons, S.; Sadler, P. J., Inorg. Chem., 2005, 44,7459-7467.)

There is therefore an ongoing need to develop new transplatinumcompounds that are effective cytotoxic and antitumor agents. It would beparticularly advantageous if the agents were water soluble, stable inthe cellular environment, and cytotoxic and antitumor active in tumorcells that develop resistance to other known anti-tumor agents.

The following also are mentioned as background:

U.S. Pat. No. 4,979,393 issued Jan. 10, 1989 to N. Farrell et al., for“Bis-platinum complexes as chemotherapeutic agents.”

U.S. Pat. No. 4,921,963 issued May 1, 1990 to Skov, Farrell, et al., for“Platinum complexes with one radiosensitizing ligand.”

U.S. Pat. No. 5,026,694 issued Jun. 25, 1991 to Skov, Farrell, et al.,for “Platinum complexes with one radiosensitizing ligand.”

U.S. Pat. No. 5,028,726 issued Jul. 2, 1991, to Farrell for “Platinumamine sulfoxide complexes.”

U.S. Pat. No. 5,107,007 issued Apr. 21, 1992 to Farrell for“Bis-platinum complexes as chemotherapeutic agents.”

U.S. Pat. No. 5,380,897 issued Jan. 10, 1995 to Hoeschele, Qu andFarrell, for “Tri(platinum) complexes.”

U.S. Pat. No. 5,409,915 issued Apr. 25, 1995 to Farrell et al., for“Bis-platinum (IV) complexes as chemotherapeutic agents.”

U.S. Pat. No. 5,624,919 issued Apr. 29, 1997 to Farrell for“Trans-platinum (IV) complexes.”

U.S. Pat. No. 5,744,497 issued Apr. 28, 1998 to Valsecchi, et al.,including Farrell, for “Trinuclear cationic platinum complexes havingantitumor activity and pharmaceutical compositions containing them.”

U.S. Pat. No. 5,770,591 issued Jun. 23, 1998 to N. Farrell for“Bis-platinum complexes as chemotherapeutic agents.”

U.S. Pat. No. 6,001,872 issued Dec. 14, 1999 to N. Farrell, et al., for“Water soluble transplatinum complexes with anti-cancer activity andmethod of using same.”

U.S. Pat. No. 6,011,166 issued Jan. 4, 2000 to Valsecchi et al.,including Farrell, for “Trinuclear cationic platinum complexes havingantitumor activity and pharmaceutical compositions containing them.”

U.S. Pat. No. 6,022,892 issued Feb. 8, 2000 to Farrell, et al., for“Bis-platinum complexes with polyamine ligands as antitumor agents.”

U.S. Pat. No. 6,060,616 issued May 9, 2000 to Farrell, et al., for“Bis-platinum complexes with polymethylene derivatives as ligands havingantitumor activity.”

U.S. Pat. No. 6,113,934 issued Sep. 5, 2000 to Farrell, et al., for“Platinum complexes with anti-viral activity and method of using same.”

U.S. Pat. No. 6,313,333 issued Nov. 6, 2001 to Da Re, et al., includingFarrell, for “Multinuclear cationic platinum complexes with antitumoractivity.”

U.S. Pat. No. 6,350,740 issued Feb. 26, 2002 to N. Farrell, for“Transplatinum complexes as cytotoxic and anticancer agents.”

S. Radulovic, Z. Tesic and S. Manic, Curr. Med. Chem., 2002, 9, 1611.

Y. Najajreh, J. M. Perez, C. Navarro-Ranninger and D. Gibson, J. Med.Chem., 2002, 45, 5189.

J. Kasparkva, O. Novakova, V. Marini, Y. Najajreh, D. Gibson, J. M.Perez and V. Brabec, J. Biol. Chem., 2003, 278, 47516.

J. Kasparkova, V. Marini, Y. Najajreh, D. Gibson and V. Brabec,Biochemistry, 2003, 42, 6312.

E. S. F. Ma, W. D. Bates, A. Edmunds, L. R. Kelland, T. Fojo and N.Farrell, J. Med. Chem., 2005, 48, 5651.

U.S. Pat. No. 6,867,316 issued Mar. 15, 2005 to Sohn, et al., for“Platinum (II) complexes of N-substituted amino dicarboxylates and thepreparation method thereof.”

U.S. Pat. Application No. 20050090478 published Apr. 28, 2005 byBarenholz, et al., for “Platinum complexes and their use in cancertreatment.”

S. Zutphen, E. Pantoja, R. Soriano, C. Soro, D. Tooke, A. Spek, H. Dulk,J. Brouwer, J. Reedijk, “New antitumour active platinum compoundscontaining carboxylate ligands in trans geometry: synthesis, crystalstructure and biological activity, Dalton Trans., 2006, 1020-1023, DOI:10.1039/b512357g (web-published Dec. 7, 2005).

SUMMARY OF THE INVENTION

The present invention provides novel, water soluble, cytotoxiccomplexes. The complexes have a trans axis that enhances solubility, yetthe complexes are relatively stable in an aqueous environment. The newPt complexes exhibit high levels of cytotoxicity. Importantly, thecomplexes are highly toxic even in cells that have become resistant tokilling by conventional Pt-based agents (e.g. cisplatin andoxaliplatin).

The invention in a preferred embodiment provides a trans-platinum IIcomplex of formula trans-[Pt(carboxylato)₂(L)(L′)] where carboxylato=aligand derived from an anion of a carboxylic acid (such as, e.g., acarboxylato ligand that is, e.g., fornate, acetate, hydroxyacetate,chloroacetate, trifluoroacetate and lactate including optical isomersthereof), and: wherein L and L′ are both planar heterocyclic amines(such as, e.g., pyridine, substituted pyridines, quinoline, subsitutedquinolines, isoquinoline, substituted isoquinolines, thiazole, andsubstituted thiazoles), or one of L or L′ is a planar heterocyclic amineand the other is not a planar heterocyclic amine and is selected fromthe group consisting of: NH₃, a branched aliphatic amine, an iminoether,a primary amine, a secondary amine and an aliphatic nitrogen-containingheterocycle, such as, e.g., trans-platinum II complexes wherein both Land L′ are planar amines; trans-platinum II complexes wherein L and L′include a planar amine and an aliphatic non-N-H₃ amine; trans-platinumII complexes wherein L is a planar amine and L′ is NH₃; trans-platinumII complexes wherein the aliphatic nitrogen-containing heterocycle isselected from the group consisting of piperzine, piperidine andpyrazole; trans-platinum II complexes wherein the carboxylato groups arein trans position to each other and L and L′ are in trans position toeach other; trans-platinum II complexes having cytotoxicity to humantumor cells (such as, e.g., trans-platinum II complexes havingcytotoxicity of at least 10 μM when measured using a standard testingmethodology for cell growth inhibition such as the MTT assay); etc.

The invention in another preferred embodiment provides a trans-platinumII complex represented by formula (I) as follows:

wherein

i) RCOO— is a carboxylate anion of a carboxylic acid and R is H or analkyl group which may be substituted or unsubstituted; and

-   -   ii) L and L′ are both planar heterocyclic amines (such as, e.g.,        pyridine, substituted pyridines, substituted quinolines,        isoquinoline, substituted isoquinolines, thiazole, and        substituted thiazoles), or

one of L or L′ is a planar heterocyclic amine and the other is selectedfrom the group consisting of NH₃, a branched aliphatic amine, animinoether, a primary amine, a secondary amine and an aliphaticnitrogen-containing heterocycle (such as, e.g.,piperazine, piperidine,pyrazole). L may be a substituent (such as, e.g., Me, Br, Cl, F).

Another embodiment of the invention provides a method of treating cancerin a patient, comprising: administering to the patient apharmaceutically effective amount of a novel trans-platinum II complexabove-mentioned.

The invention in another preferred embodiment provides a method ofkilling cancer cells, comprising: contacting cancer cells with a noveltrans-platinum II complex above-mentioned.

Also in a preferred embodiment the invention provides a method ofsolubilizing a trans-platinum compound to be water soluble, comprisingattaching at least one solubilizing agent to said compound, wherein thesolubilizing agent comprises a first carboxylato group and a secondcarboxylato group positioned trans to one another in said compound,wherein the carboxylate groups are the same, such as, e.g., methodswherein said compound is a trans-platinum II complex of formulatrans-[Pt(carboxylato)₂(L)(L′)] where carboxylato=a ligand (e.g.,formate, acetate, hydroxyacetate, chloroacetate, trifluoroacetate andlactate including optical isomers thereof) derived from an anion of acarboxylic acid, and wherein L and L′ are both planar heterocyclicamines (e.g., pyridine, substituted pyridines, substituted quinolines,isoquinoline, substituted isoquinolines, thiazole, and substitutedthiazoles), or one of L or L′ is a planar heterocyclic amine and theother cannot be a planar heterocylic amine and is selected from thegroup consisting of: NH₃, a branched aliphatic amine, an iminoether, aprimary amine, a secondary amine and an aliphatic-containing heterocycle(e.g., piperazine, piperidine, pyrazole).

The invention in another preferred embodiment provides a method ofsynthesizing a trans-platinum II complex, comprising at least the stepsof: converting a starting material selected from the group consisting ofK₂PtCl₄ and cis-[PtCl₂(NH₃)₂] into an intermediate trans diido compoundtrans-[Pt I₂(L)(L′)], wherein L is selected from the group consisting ofpyridine, quinoline, isoquinoline and thiazole; L′ is NH₃; followed byconverting the intermediate trans diido compound into a trans-platinumII complex trans-[Pt(RCOO)₂(L)(NH₃)]; such as, e.g., synthesis methodsincluding synthesis of a symmetric (L=L′) trans platinum II complex froma starting material K₂[PtCl₄]; synthesis methods including synthesis ofan unsymmetric (L and L′ differ) trans platinum II complex from astarting material cis-[PtCl₂(L)₂] or cis-[PtCl₂(L′)₂]; synthesis methodsincluding reacting the starting material with L and L′ to substitute thePt with L and L′, followed by a step of reacting the L, L′ substitutedPt compound with H Cl to form a trans-[Pt(Cl)₂(L)(L′)] compound,followed by a step of reacting the trans-[Pt(Cl)₂(L)(L′)] compound withNaI in acetone or a solvent other than DMF to form the diidointermediate compound; synthesis methods including reacting the diiodointermediate compound with Ag(RCOO) in acetone or a solvent other thanDMF until trans-[Pt(RCOO)₂(L)(L′)] is formed; synthesis methodsincluding forming the trans platinum (II) complex from the startingmaterial without using DMF in any step; etc.

In another preferred embodiment, the invention provides a method ofsynthesizing a trans-platinum II complex, comprising:

converting a starting material selected from the group consisting ofK₂[PtCl₄] and cis-[PtCl₂(NH₃)₂] into a trans-platinum IR complextrans-[Pt(RCOO)₂(L)(L′)] without using DMF nor any high-boiling solventwith a boiling point in a range of DMF in any step; such as, e.g.,synthesis methods including a step of forming a trans-[Pt(RCOO)₂(L)(L′)]via a diido intermediate compound.

The invention in a further preferred embodiment provides a method ofreleasing a hydrolyzed trans-platinum compound comprising: selecting acarboxylato group for a desired rate of release associated with thecarboxylato group for a hydrolyzed species of a trans-platinum compound;attaching at least two carboxylato groups trans to one another to thetrans-platinum compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Comparison of Resistance Factors of a representativeisostructural TPA chloride and acetate complex in cisplatin andoxaliplatin-resistant (1AP CP80 and 1A9 OX40 respectively) ovarian tumorcell lines. Assays performed as per ref. 39 and 40.

FIG. 2. TPA carboxylate compounds studied for Example 6.

FIG. 3. Initial hydrolysis rate constants determined from HPLCexperiments in H₂O at 37° C.

FIG. 4. Cytotoxicity in A2780 Human Ovarian Cells of the TPA carboxylatecompounds.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention relates to new trans-platinum II complexes withcytotoxic activity. Significantly, these complexes are cytotoxic intumor cells that are resistant to the toxic effects of traditionalPt-based anti-tumor drugs such as cisplatin and oxaliplatin. Withoutbeing bound by theory, it appears that, unlike the currently availableplatinum drugs, these complexes do not produce DNA damage by intrastrandcross-linking of base pairs. Instead, they may cause cell death bycausing a set of structurally distinct DNA lesions including DNA-proteincrosslinks. As a result, tumor cells that have developed resistance toknown platinum drugs are still sensitive to these trans compounds.Current FDA approved platinum drugs include cisplatin, carboplatin andoxaliplatin, all of which have a cis geometry around the platinum atom.It is expected that these new trans compounds will complement existingchemotherapeutic agents, although they may also be used to kill anytumor cell, regardless of whether or not the tumor cell is resistant toclassical Pt-based anti-tumor agents.

Remarkably, the complexes, which contain two carboxylate ligands intrans position, are very water soluble in comparison to the parentdichlorides, and yet are kinetically inert to hydrolysis and thus stablein an aqueous environment.

These compounds have the general formula trans [Pt(carboxylato)₂(L)(L′)]where “carboxylato”is an anionic ligand derived from a carboxylic acid,for example, formate, acetate, hydroxyacetate, chloroacetate,trifluoroacetate, lactate, and the like; L is preferably a planar,aromatic amine like pyridine, quinoline, isoquinoline or thiazole; andL′ can be NH₃ or the same amine as L. Other examples of L will alsoinclude aliphatic cyclic amines (such as cyclohexylamine, piperazine,piperidine), branched aliphatic amines and iminoethers. The compoundsthus represent the first cytotoxic transplatinum compounds with N₂O₂donor sets, similar to carboplatin and oxaliplatin.

Without being bound by theory, it is probable that the reactivity ofthese compounds is dictated mostly by the ability of the labile ligands(the carboxylato leaving groups) to be replaced by water producingaquated (hydrolysed) species, while the solubility is enhanced by thepresence of a trans axis and the hydrophilicity of both the leavinggroup and the aromatic amine. The inclusion of polar groups in theligands enhances the solubility while strong electronegative groupsenhance the leaving group character.

The Pt(II) based complexes of the invention are schematically depictedin Formula I.

Herein, “planar heterocyclic amine” refers, e.g., to pyridine,substituted pyridines, substituted quinolines, isoquinoline, substitutedisoquinolines, thiazole, and substituted thiazoles.

In “RCOO—” which has been mentioned for practicing the invention and isa carboxylate anion of a carboxylic acid, R is, for example, H, CH₃, ora straight or branched alkyl chain of from about 2 to about 5 carbons,and may be substituted or unsubstituted (e.g., with OH, Cl, F, Me, Br,etc., of which preferred substituents are Me, Br, Cl and F). In otherwords, the “OOCR” carboxylato moiety represents a carboxylic acid suchas, for example, formate, acetate, hydroxyacetate, chloroacetate,trifluoroacetate, lactate (in the form of either optical isomer), etc. Rmay be substituted or unsubstituted.

An example of an inventive compound according to Formula I is a compoundin which L is pyridine, quinoline or isoquinoline, or another N-donorligand (e.g., cyclic aliphatic amines, linear and branched-chain alkylamine and iminoethers) and L is not NH₃, and in which L′ is pyridine,quinoline, isoquinoline or thiazole or an N-donor ligand (e.g., cyclicaliphatic amines, linear and branched-chain alkyl amine andiminoethers), or NH₃. L and L′ may be the same OF different.

The invention also provides other trans-platinum (II) complexes,comprising: an N₂O₂ donor set, preferably trans-platinum (II) complexeswherein the N₂O₂ donor set comprises a donor (neutral) ligand N and aleaving (anionic) group O.

The invention also provides pharmaceutical compositions containing thecompounds of formula I with pharmaceutically acceptable excipients.While the compounds can be administered in the pure form, morefrequently they are administered in a pharmaceutically acceptableformulation including suitable elixirs, binders, and the like or aspharmaceutically acceptable salts or other derivatives. It should beunderstood that the pharmaceutically acceptable formulations and saltsinclude liquid and solid materials conventionally utilized to prepareinjectable dosage forms and solid dosage forms such as tablets andcapsules. Water may be used for the preparation of injectablecompositions which may also include conventional buffers and agents torender the injectable composition isotonic. Other potential additivesinclude: colorants; surfactants (TWEEN, oleic acid, etc.); and bindersor encapsulants (lactose, liposomes, etc). Solid diluents and excipientsinclude lactose, starch, conventional disintergrating agents, coatingsand the like. Preservatives such as methyl paraben or benzalkiumchloride may also be used. Depending on the formulation, it is expectedthat the active composition will consist of 1-99% of the Pt complexesand the vehicular “carrier” will constitute 1-99% of the composition.The pharmaceutical compositions of the present invention may include anysuitable pharmaceutically acceptable additives or adjuncts to the extentthat they do not hinder or interfere with the therapeutic effect desiredof the Pt complex.

The invention also provides a method of treating tumors which aresusceptible to platinum complex treatment with one or more compounds offormula I. Implementation of the claimed method will generally involveidentifying patients suffering from tumors and administering theplatinum compound(s) in an acceptable form by an appropriate route. Thedosage to be administered is usually determined in Phase I clinicaltrials and may vary depending on the age, gender, weight and overallhealth status of the individual patient, as well as the nature of thecancer itself, among other considerations. Administration can be oral orparenteral, including intravenously, intramuscularly, subcutaneously,etc., or by other routes (e.g. transdermal, sublingual, aerosol, etc.).The administration of pharmaceutical compositions of the presentinvention can be intermittent, or at a gradual or continuous, constantor controlled rate to a patient. In addition, the time of day and thenumber of times per day that the pharmaceutical formulation isadministered can vary. Further, the effective dose can vary dependingupon factors such as the mode of delivery, gender, age, and otherconditions of the patient, as well as tumor type and stage or grade.

Generally, for parenteral administration in humans, dosages in the rangeof from about 0.1 to about 500 mg active Pt compound/kg body weight/24hr., more preferably 1.0 to 20.0 active Pt compound/kg body weight/24hr., are effective. The level of efficacy and optimal amount of dosagefor any given Pt complex varies from complex to complex.

The Pt complexes of the present invention may be administered alone orin combination with other cancer therapies, e.g. other antitumor agents,radiation, etc. Since the compounds of the present invention arecomplementary to known anti-tumor cis-Pt compounds and are toxic tocancer cells that are resistant to known compounds, one strategy forcancer treatment is to use the drugs of the present invention incombination with traditional cis-Pt drugs. For example, administrationof the two genres of drugs may be alternated in a patient, or evenadministered together. Alternatively, it may be desirable to wait untilresistance to the conventional cis-Pt drugs is evident and then begintreatment with the novel trans-Pt drugs. Yet another alternative is totreat first with the trans-Pt drugs of the invention, and follow thattreatment with conventional drugs if resistance to trans-Pt drugsdevelops. In addition, more than one of the novel Pt-complexes describedherein may be administered to a patient, either separately, or in asingle preparation. All such variations, as well as others that willoccur to those of skill in the art, are encompassed by the methods ofthe present invention.

The invention further provides a process to prepare compounds of formulaI. When L and L′ are the same, the preparation generally involves thesteps of reacting K₂PtCl₄ with excess nitrogen donor ligand, L, underconditions in which the ligand replaces the halogen ion of the salt toeventually form the cation [Pt(L)₄]²⁺ which is then reacted inconcentrated HCl under conditions in which trans-[PtCl₂(L₂)] is formed.The trans-[PtCl₂(L₂)] is then either:

-   1) preferably, reacted with excess NaI under conditions where I    replace Cl, forming trans-[PtI₂(L₂)] and the trans-[PtI₂(L₂)]    subsequently reacted with Ag(RCOO) under conditions in which I is    replaced by RCOO to form trans-[Pt(COOR₂)(L₂)]; or-   2) alternately, reacted with Ag(RCOO) under conditions in which Cl    is is replaced by RCOO to form trans-[Pt(COOR₂)(L₂)].

In the case of 1) the critical trans-[PtI₂(L₂)] may also be prepareddirectly from the [Pt(L)₄]²⁺ cation and I. When L and L′ are not thesame and L′=N₃, the procedure is the same except the starting materialis cis-[Pt(Cl₂)(NH₃)₂] instead of K₂PtCl₄. When L and L′ are not thesame the procedure is the same except the starting material iscis-[Pt(Cl₂)(L)₂] or cis-[Pt(Cl₂)(L′)₂] and the intermediate cation is[Pt(L)₂(L′)₂]²⁺ which is converted into trans-[PtI₂(L)(L′)] as above.The synthesis of exemplary Pt II complexes of the invention is describedin detail in Example 6 below.

Non-limiting examples of novel compounds according to this invention areas follows:

A trans-platinum(II) complex wherein the N₂O₂ donor set comprises adonor (neutral) ligand N and a leaving (anionic) carboxylato group O;

A trans-platinum(II) complex of formula trans-[Pt(carboxylato)₂(L)(L′)]where carboxylato=an anionic ligand of a carboxylic acid (with examplesof L being, e.g., a planar amine; an aliphatic cyclic amine (such as,e.g., cyclohexylamine; piperazine, piperidine (which may be substitutedor unsubstituted); etc.); a branched aliphatic amine; an iminoether;etc.);

A trans-platinum (II) complex including a carboxylato acid ligandselected from the group consisting of formate, acetate, hydroxyacetate,chloroacetate, trifluoroacetate and lactate (in either optical isomericform of lactate);

A trans-platinum (II) complex comprising two carboxylato groups disposedin trans position from each other about a platinum atom, wherein the twocarboxylato groups are the same;

A trans-platinum(II) complex represented by a formulatrans-[PtX₂(L)(L′)] wherein the X groups are in trans position from eachother and L and L′ are in trans position from each other, X is an anionof a carboxylic acid; L and L′ (which may be the same or different)together form an N₂O₂ donor set in which L is a heterocyclic aromaticamine or an N-donor ligand other than NH₃ and L′ is a heterocyclicaromatic amine or an N-donor ligand and may include NH₃.

Some exemplary inventive compounds include, e.g., the following, by wayof non-limiting limiting examples:

-   trans-[Pt(OAc)₂(NH₃)(py)]    (trans-[diacetato(ammine)(pyridine)platinum(II)]);-   trans-[Pt(OAc)₂(NH₃)(2Me-py)]    (trans-[diacetato(ammine)(2-methylpyridine)platinum(II)]);-   trans-[Pt(OAc)₂(NH₃)(3Me-py)]    (trans-[diacetato(ammine)(3-methylpyridine)platinum(II)]);-   trans-[Pt(OAc)₂(NH₃)(4Me-py)]    (trans-[diacetato(ammine)(4-methylpyridine)platinum(II)]);-   trans-[Pt(OAc)₂(NH₃)(tz)]    (trans-[diacetato(ammine)(thiazole)platinum(II)]);-   trans-[Pt(OAc)₂(NH₃)(quin)]    (trans-[diacetato(ammine)(quinoline)platinum(II)]);-   trans-[Pt(OAc)₂(NH₃)(Iq)]    (trans-[diacetato(ammine)(isoquinoline)platinum(II)]);-   trans-[Pt(OFm)₂(NH₃)(py)]    (trans-[diformato(ammine)(pyridine)platinum(II)]);-   trans-[Pt(OFm)₂(NH₃)(2Me-py)]    (trans-[diformato(ammine)(2-methylpyridine)platinum(II)]);-   trans-[Pt(OFm)₂(NH₃)(3Me-py)]    (trans-[diformato(ammine)(3-methylpyridine)platinum(II)]);-   trans-[Pt(OFm)₂(NH₃)(4Me-py)]    (trans-[diformato(ammine)(4-methylpyridine)platinum(II)]);-   trans-[Pt(OFm)₂(NH₃)(tz)]    (trans-[diformato(ammine)(thiazole)platinum(II)]);-   trans-[Pt(OFm)₂(NH₃)(quin)]    (trans-[diformato(ammine)(quinoline)platinum(II)]);-   trans-[Pt(OFm)₂(NH₃)(Iq)]    (trans-[diformato(ammine)(isoquinoline)platinum(II)]);-   trans-[Pt(lact)₂(NH₃)(py)]    (trans-[dilactato(ammine)(pyridine)platinum(II)]);-   trans-[Pt(lact)₂(NH₃)(2Me-py)]    (trans-[dilactato(ammine)(2-methylpyridine)platinum(II)]);-   trans-[Pt(lact)₂(NH₃)(3Me-py)]    (trans-[dilactato(ammine)(3-methylpyridine)platinum(II)]);-   trans-[Pt(lact)₂(NH₃)(4Me-py)]    (trans-[dilactato(ammine)(4-methylpyridine)platinum(II)]);-   trans-[Pt(lact)₂(NH₃)(tz)]    (trans-[dilactato(ammine)(thiazole)platinum(II)]);-   trans-[Pt(lact)₂(NH₃)(quin)]    (trans-[dilactato(ammine)(quinoline)platinum(II)]);-   trans-[Pt(lact)₂(NH₃)(Iq)]    (trans-[dilactato(ammine)(isoquinoline)platinum(II)]);-   trans-[Pt(OAcOH)₂(NH₃)(py)]    (trans-[dihydroxyacetato(ammine)(pyridine)platinum(II)]);-   trans-[Pt(OAcOH)₂(NH₃)(2Me-py)]    (trans-[dihydroxyacetato(ammine)(2-methylpyridine)platinum(II)]);-   trans-[Pt(OAcOH)₂(NH₃)(3Me-py)]    (trans-[dihydroxyacetato(ammine)(3-methylpyridine)platinum(II)]);-   trans-[Pt(OAcOH)₂(NH₃)(4Me-py)]    (trans-[dihydroxyacetato(ammine)(4-methylpyridine)platinum(II)]);-   trans-[Pt(OAcOH)₂(NH₃)(tz)]    (trans-[dihydroxyacetato(ammine)(thiazole)platinum(II)]);-   trans-[Pt(OAcOH)₂(NH₃)(quin)]    (trans-[dihydroxyacetato(ammine)(quinoline)platinum(II)]);-   trans-[Pt(OAcOH)₂(NH₃)(Iq)]    (trans-[dihydroxyacetato(ammine)(isoquinoline)platinum(II)]);-   trans-[Pt(TFA)₂(NH₃)(py)]    (trans-[bis(trifluoro)acetato(ammine)(pyridine)platinum(II)]);-   trans-[Pt(TFA)₂(NH₃)(2Me-py)]    (trans-[bis(trifluoro)acetato(ammine)(2-methylpyridine)platinum(II)]);-   trans-[Pt(TFA)₂(NH₃)(3Me-py)]    (trans-[bis(trifluoro)acetato(ammine)(3-methylpyridine)platinum(II)]);-   trans-[Pt(TFA)₂(NH₃)(4Me-py)]    (trans-[bis(trifluoro)acetato(ammine)(4-methylpyridine)platinum(II)]);-   trans-[Pt(TFA)₂(NH₃)(py)]    (trans-[bis(trifluoro)acetato(ammine)(thiazole)platinum(II)]);-   trans-[Pt(TFA)₂(NH₃)(quin)]    (trans-[bis(trifluoro)acetato(ammine)(quinoline)platinum(II)]);-   trans-[Pt(TFA)₂(NH₃)(Iq)]    (trans-[bis(trifluoro)acetato(ammine)(isoquinoline)platinum(II)]);-   trans-[Pt(OAc)₂(py)₂] (trans-[diacetatobis(pyridine)platinum(II)]);-   trans-[Pt(OAc)₂(2Me-py)₂]    (trans-[diacetatobis(2-methylpyridine)platinum(II)]);-   trans-[Pt(OAc)₂(3Me-py)₂]    (trans-[diacetatobis(3-methylpyridine)platinum(II)]);-   trans-[Pt(OAc)₂(4Me-py)₂]    (trans-[diacetatobis(4-methylpyridine)platinum(II)]);-   trans-[Pt(OAc)₂(tz)₂] (trans-[diacetatobis(thiazole)platinum(II)]);-   trans-[Pt(OFm)₂(py)₂] (trans-[diformatobis(pyridine)platinum(II)]);-   trans-[Pt(OFm)₂(2Me-py)₂]    (trans-[diformatobis(2-methylpyridine)platinum(II)]);-   trans-[Pt(OFm)₂(3Me-py)₂]    (trans-[diformatobis(3-methylpyridine)platinum(II)]);-   trans-[Pt(OFm)₂(4Me-py)₂]    (trans-[diformatobis(4-methylpyridine)platinum(II)]);-   trans-[Pt(OFm)₂(tz)₂] (trans-[diformatobis(thiazole)platinum(II)]);-   trans-[Pt(lact)₂(py)₂] (trans-[dilactatobis(pyridine)platinum(II)]);-   trans-[Pt(lact)₂(2Me-py)₂]    (trans-[dilactatobis(2-methylpyridine)platinum(II)]);-   trans-[Pt(lact)₂(3Me-py)₂]    (trans-[dilactatobis(3-methylpyridine)platinum(II)]);-   trans-[Pt(lact)₂(tz)₂] (trans-[dilactatobis(thiazole)platinum(II)]);-   trans-[Pt(OAcOH)₂(py)₂]    (trans-[dihydroxyacetatobis(pyridine)platinum(II)]);-   trans-[Pt(OAcOH)₂(2Me-PY)₂]    (trans-[dihydroxyacetatobis(2-methylpyridine)platinum(II)]);-   trans-[Pt(OAcOH)₂(3Me-PY)₂]    (trans-[dihydroxyacetatobis(3-methylpyridine)platinum(II)]);-   trans-[Pt(OAcOH)₂(4Me-py)₂]    (trans-[dihydroxyacetatobis(4-methylpyridine)platinum(II)]);-   trans-[Pt(OAcOH)₂(tz)₂]    (trans-[dihydroxyacetatobis(thiazole)platinum(II)]);-   trans-[Pt(TFA)₂(py)₂]    (trans-[bis(trifluoro)acetatobis(pyridine)platinum(II)]);-   trans-[Pt(TFA)₂(2Me-py)₂]    (trans-[bis(trifluoro)acetatobis(2-methylpyridine)platinum(II)]);-   trans-[Pt(TFA)₂(3Me-py)₂]    (trans-[bis(trifluoro)acetatobis(3-methylpyridine)platinum(II)]);-   trans-[Pt(TFA)₂(4Me-py)₂]    (trans-[bis(trifluoro)acetatobis(4-methylpyridine)platinum(II)]);-   trans-[Pt(TFA)₂(tz)₂]    (trans-[bis(trifluoro)acetatobis(thiazole)platinum(II)]),-   with the following definitions applying to the above compounds:    OAc=acetate CH₃COO⁻;-   OFm=formate HCOO⁻; Lact=lactate CH₃C*H(OH)COO⁻(*=R or S chirality);    OAcOH=hydroxyacetate HOCH₂COO⁻; TFA=trifluoroacetate CF₃COO¹;    py=pyridine; 2Me-py=2-methylpyridine; 3Me-py=3-methylpyridine;    4Me-py=4-methylpyridine; tz=thiazole; Iq=isoquinoline; and    quin=quinoline.

Novel trans-platinum II complexes having cytotoxicity to human tumorcells are preferred, such as trans-platinum II complexes havingcytotoxicity measured of at least 10 μM when measured using a MTT assayor other standard testing methodology for cell growth inhibition.

EXAMPLES Examples 1-4 Synthesis of Trans-platinum compounds of generalformula trans-[Pt(carboxylato)₂(L)(L′)]

The synthesis of trans-platinum compounds of general formula transPt(carboxylato)₂(L)(L′) is shown in Scheme 1. A general syntheticprocedure was developed that would lead to either symmetric orunsymmetric complexes depending on the starting

that would lead to either symmetric or unsymmetric complexes dependingon the starting material. When a symmetric compound is desired, thestarting reagent used is K₂ PtCl₄, and when a non-symmetric compound isrequired the starting reagent is cis-DDP, cis-Pt(NH₃)₂ Cl₂.

A suspension or solution of the starting platinum reagent in water isreacted with an excess of the aromatic amine, L, under reflux for 1.5hr. The solution is removed from heat, activated charcoal is added, andthe mixture is stirred for 10 min. After the solution is filtered,excess concentrated HCl is added and the mixture heated under reflux for6 hr during which time a yellow precipitate is formed. After cooling themixture overnight, the yellow product is filtered, washed with water,ethanol, and ethyl ether, and dried in vacuo. The product,trans-[PtCl₂LL′] can then be dissolved in DMF with warming and added toa mixture of 2.1 equiv. of the silver salt of the carboxylic acid inH2O. The mixture is stirred in the dark at 50° C. for 2 days. Theprecipitate of AgCl is filtered off and the filtrate is evaporated todryness under vacuum. The residue is redissolved in hot methanol andleft to crystallize.

A modification of this procedure involves the activation of the leavinggroups in the precursor trans-[PtCl₂LL′] by replacing the chloride atomswith iodide atoms. Higher yields and cleaner products can sometimes beobtained using this approach. If activation is desired, the compound issuspended in a solution of excess NaI in acetone and the mixture stirreda room temperature overnight. NaCl is filtered off and the filtrateevaporated to dryness. The residue, trans-[PtI₂LL′] is washed with warmH₂O. methanol and ethyl ether and dried in vacuo. This compound isdissolved or suspended in acetone and added to 2.1 equiv. of the silversalt of the appropriate carboxylate. The mixture is stirred in the darkat 40-50° C. for 24 hr. AgI is filtered off through a pad of celite andthe filtrate is concentrated until a white precipitate starts to appearat which point the mixture is placed in the fridge for a few hours. Theprecipitate is filtered, washed with ethyl ether, and dried in vacuo.The product usually can be recrystallized from methanol.

Silver salts of small, water soluble carboxylic acids can be prepared byreacting excess of the carboxylic acid with a suspension of silvercarbonate or silver oxide in water. The mixture is stirred for 2 h inthe dark at room temperature. The solution is filtered, concentrateduntil a precipitate appears and stored in the fridge to crystallize. Thecrystals are filtered, washed with cold water, and dried in vacuo in thedark. A second crop can be obtained when the filtrate is furtherconcentrated.

Example 1 trans-PtCl₂(py)₂ Synthesis

A solution of 2.00 g (4.81 mmol) of K₂ PtCl₄ and 6.00 mL (74.2 mmol) ofpyridine in 160 mL of water was stirred at reflux. After 1 h, 10 mL ofconcentrated HCl were added to the solution and heating was continuedfor 5 h. After the mixture is stored at 4 ° C. for 12 h, the lightyellow precipitate was filtered off, washed with hot water, ethanol anddiethyl ether, and finally dried in vacuo. Yield: 1.95 g (95%). ¹H NMRin DMSO-d6: 8.79 ppm (2H d), 8.02 ppm (1H dt), 7.54 ppm (2H t).

Example 2 trans-[Pt(OAcOH)₂(py)₂] Synthesis

trans-PtCl₂(py)2, 400 mg, 0.943 mmol, was dissolved in 20 mL DMF bywarming to 50° C. with stirring. Silver hydroxyacetate, 345 mg, 1.89mmol was dissolved in 20 mL of warm water and added to the yellowsolution of trans-PtCl₂(py)₂. The mixture was heated to 50° C. andstirring continued in the dark for 2 days. The mixture was filtered togive a yellow solution and evaporated to dryness to give 488 mg of ayellow residue. The residue was dissolved almost completely in methanoland the material that remained undissolved was filtered off. Thefiltrate was concentrated and placed in the fridge to crystallize.Yield: 147 mg (31%). Anal. Calcd. for C₁₄H₁₆N₂O₆Pt: % C, 33.41; % H,3.20; % N, 5.57. Found: % C, 33.25; % H 2.89; % N, 5.52. ¹H NMR in D₂O:8.514 ppm (2H d), 7.995 ppm (1H tt), 7.514 ppm (2H t), 4.020 ppm (2H s).¹⁹⁵Pt NMR in D₂O: −1459 ppm.

Example 3 trans-[Pt(OAc)₂(NH₃)(iquin)] Synthesis

AgOAc (700 mg, 4.19 mmol) was added to a solution oftrans-[PtI₂(NH₃)(iquin)] (1.0 g, 1.68 mmol) in 225 mL acetone. After themixture was stirred in the dark for 24 h at ambient temperature, 300 mLof acetone was added. The reaction mixture was heated to reflux for 15min until the product dissolved dissolved. AgI and unreacted AgOAc werefiltered off through a pad of Celite while still hot. The clear andcolorless filtrate was concentrated to 50 mL and placed in the fridge(5° C.) for 20 h giving a off-white precipitate. The product wasfiltered off, washed with Et₂O (2×10 mL) and dried in vacuo. Theproduct, which may contain minute amounts of Ag species, wasrecrystallized from acetone (100 mg/60 mL). Yield: 0.585 g (76%). Anal.Calcd for C₁₃H₁₆N₂O₄Pt.H₂O: C, 32.71;

H, 3.80; N, 5.87. Found: C, 32.44; H, 3.41; N, 5.75. ¹H NMR in D₂O: 9.14ppm (1H s), 8.24 ppm (1H d), 8.11 ppm (1H d), 7.98 ppm (1H d), 7.90 ppm(1H t), 7.84 ppm (1H d), 7.76 ppm (1H t), 1.96 ppm (6H s). ¹⁹⁵Pt NMR inD₂O:1430 ppm.

Example 4 trans-[Pt(OAc)₂(NH₃)(iquin)] Synthesis

A suspension of AgOAc (162 mg, 0.97 mmol) in 12 mL H₂O which has beenheated at 60° C. for 10 min in the dark is added to a solution oftrans-[PtCl₂(NH₃)(iquin)] (200 mg, 0.49 mmol) in 10 mL DMF. The mixtureis stirred in the dark at 50° C. for 2 days. AgCl is filtered offthrough a pad of Celite and washed with MeOH. The combined yellowfiltrates are evaporated to dryness under vacuum and the yellow residueis redissolved in MeOH (15 mL). The yellow solution is treated withactivated charcoal (200 mg) for 30 min. The mixture is passed through anew pad of Celite and the pale yellow filtrate is evaporated to dryness.The residue is redissolved in minimal MeOH (5 mL) with slight heating(60° C.). The solution is stored at 5° C. for 3 h during which time paleyellow crystals formed. The product is filtered off, washed with Et₂O(10 mL) and dried in vacuo. Yield: 50% pale yellow solid. Anal. Calcd.for C₁₃H₁₆N₂O₄Pt.(H₂O): % C, 32.71; % H, 3.80; % N 5.87. Found: % C,32.44; % H, 3.42; % N, 5.75. ¹H NMR in D₂O: 9.14 ppm (1H s), 8.24 ppm(1H d), 8.11 ppm (1H d), 7.98 ppm (1H d), 7.90 ppm (1H t), 7.84 ppm (1Hd), 7.76 ppm (1H t), 1.96 ppm (6H s). ¹⁹⁵Pt NMR in D₂O: −1429 ppm.

Using the previous methods, the following selected compounds were alsoprepared:

t-[Pt(OAcOH)₂(py)₂], Anal. Calcd. for C₁₄H₁₆N₂O₆Pt: C, 33.41; H, 3.20;N, 5.57. Found: C, 33.25; H 2.89; N, 5.52. ¹⁹⁵Pt NMR in D₂O: −1459 ppm.¹H NMR in D₂O: 8.514 ppm (2H d), 7.995 ppm (1H tt), 7.514 ppm (2H t),4.020 ppm (2H s).

t-[Pt(lactate)₂(py)₂], Anal. Calcd. for C₁₆H₂₀N₂O₆Pt: C, 36.16; H, 3.79;N, 5.27. Found: C, 36.20; H, 3.49; N, 5.20. ¹H NMR in D₂O: 8.51 ppm (2Hbd), 8.00 ppm (1H tt), 7.52 ppm (2H bt), 4.18 ppm (1H q), 1.11 ppm (3Hd). ¹⁹⁵Pt NMR in D₂O: −1481 ppm.

t-[Pt(CF₃COO)₂(py)₂], Anal. Calcd. for C₁₄H₁₀F₆N₂O₄Pt: C, 29.03; H,1.74; N, 4.84. Found: C, 28.95; H, 1.38; N, 4.78. ¹⁹⁵Pt NMR in CD₃OD:−1431 ppm. ¹H NMR in CD₃OD: 8.651 ppm (2H bd), 8.042 ppm (1H tt), 7.582ppm (2H bt). ¹⁹F NMR in CD₃OD: −76.39 ppm.

t-[Pt(CH₂ClCOO)₂(py)₂], Anal. Calcd. for C₁₄H₁₄Cl₂N₂O₄Pt: C, 31.12; H,2.61; N, 5.19. Found: C, 31.26; H, 2.40; N, 5.08. ¹H NMR in DMSO-d6:8.57 ppm.(2H bd), 8.05 ppm (1H tt), 7.57 ppm (2H bt), 4.06 ppm (2H s).

t-[Pt(NH₃)(lactato)₂(Iq)], Anal. Calcd. for C₁₅H₂₀N₂O₆Pt: C, 34.69; H,3.88; N, 5.39. Found: C, 33.23; H, 3.55; N, 5.18. ¹H NMR in D₂O: 9.15ppm (1H s), 8.24 ppm (1H d), 8.125 ppm (2H s), 7.98 ppm (1H s), 7.91 ppm(1H t), 7.86 ppm (1H d), 7.77 ppm (1H t), 4.23 ppm (2H q), 1.17 ppm (3Hd). ¹⁹⁵Pt NMR in D₂O: −1466 ppm.

t-[Pt(NH₃)(HCOO)₂(Iq)], Anal. Calcd. for C₁₁H₁₂N₂O₄Pt: C, 30.63; H,2.80; N, 6.50. Found: C, 29.46; H, 3.04; N, 6.23. ¹H NMR in D₂O: 9.18ppm (1H s), 8.26 ppm (1H d), 8.11 ppm (1H d), 7.98 ppm (1H d), 7.90 ppm(1H t), 7.84 ppm (1H d), 7.77 ppm (1H t), 7.43 ppm (2H s). ¹⁹⁵Pt NMR inD₂O: −1450 ppm.

Example 5 Enhancement of Aqueous Solubility and Stability Employing aTrans Acetate Axis in Transplanar Amine Platinum Compounds whileMaintaining the Biological Profile Background/Comparative Examples

Since the discovery of its anticancer activity cisplatin(cis-[PtCl₂(NH₃)₂], cis-DDP) has developed into a mainstay of manychemotherapeutic regimens. ^(1,2,3) The structure-activity relationships(SARs) developed since the introduction of cisplatin into the clinicemphasized the necessity for leaving groups having a cis geometry and anoverall neutral charge on the Pt agent. Although many permutations basedon this chemotype (cis-[Pt(amine)₂X₂], neutral Pt entities) have beenexplored, to date only carboplatin (cis-[Pt(CBDCA)(NH₃)₂],(CBDCA=cyclobutane-1,1-dicarboxylate) and oxaliplatin ([Pt(ox)(dach)](dach=1,2-diaminocyclohexane, ox=oxalato) have gained world-wideacceptance in the clinic.⁴

The principal factors affecting platinum complex cytotoxicity, valid forboth inherent and acquired resistance, are (a) cellular uptake andefflux; (b) the nature and structure of target Pt-DNA adducts and (c)the extent of metabolizing reactions with sulfur nucleophiles, generallyconsidered to be deactivating. One approach to expand the anticancerspectrum of platinum agents has been to examine structurally uniqueplatinum agents with the hypothesis that novel Pt-DNA adducts notaccessible to cisplatin may result in a differential cellular response.⁵One such class is represented by polynuclear platinum compounds, asexemplified by the trinuclear BBR3464, where the results of Phase IIclinical trials showed partial responses in cisplatin-relapsed ovariancancer.^(6,7) A second approach has been to explore the trans geometry.^(8,9)The paradigm for the early SARs was that the trans geometry,trans-[PtCl₂(NH₃)₂] (trans-DDP), was therapeutically inactive. A numberof factors may contribute to this difference between simple geometricisomers. The trans isomer is kinetically more reactive than the cisisomer which may contribute to its deactivation. In addition, theprimary toxic lesion in DNA formed by cis-DDP, a 1,2-intrastrandcross-link between adjacent GG base pairs, is sterically inaccessible tothe trans isomer. The minor interstrand crosslink formed by cis-DDP isbetween adjacent GG base pairs in (GC) sequences. ^(10,11)In contrast,the trans isomer forms a unique 1,1-interstrand cross-link between GN7and CN3 of the same GC base pair, a lesion distinctly less distorting toDNA structure than the cis case. ¹² Protein recognition of both types ofcrosslink, and the biological consequences thereof, is likely to besignificantly different between the two isomers.^(13,14)

Substitution of the NH₃ group by a sterically hindered planar aminetrans-[PtCl₂(L)(L′)] (L=NH₃, L′=pyridine, quinoline, thiazole, etc.and/or L=L′=pyridine or thiazole) gives transplanaramine (TPA) compoundswith cytotoxicity comparable to cisplatin in human tumor cell lines.^(15.16)Use of the planar amine enhances cytotoxicity up to 100-foldover trans-[PtCl₂(NH₃)₂]. Further, the compounds generally maintaincytotoxicity in cisplatin-resistant lines. Since our initial reports, anumber of other groups have confirmed the effects of a stericallydemanding group in modulation of the cytotoxicity of the transplatinumstructure—amines used include cyclohexylamine,¹⁷ branched aliphaticamines,¹⁸ piperazine, piperidine^(19,20) and iminoethers.⁹ In generalthe cytotoxicity of these various compounds is in the 1-20 μM range andis characterized by lack of cross-resistance to cisplatin. The DNAbinding profiles of these various compounds show a wide diversity incomparison to those of the cisplatin family²¹ and it is important toexamine the chemistry and profile of DNA binding of these newtransplatinum compounds and place them in context.

Despite the wide variation of amine carrier ligands, there has beenlittle in vivo antitumor activity reported thus far for thetransplatinum geometry.^(8,22)

All previously-reported modifications of transplatinum compounds are ofthe form trans-[PtCl₂(L)(L′)] where L and L′ are various amines otherthan NH₃. These comparative compounds are poorly soluble in aqueousmedium and still contain the relatively reactive Cl—Pt—Cl axis. Toaddress the poor solubility of the comparative compounds, complexes wereexamined wherein a trans axis that enhances aqueous solubility isutilized. One approach was to develop trans-Pt compounds containing aplanar amine chelating moiety (such as [SP-4-2]-[PtCl(NH₃)(pyOAc—N,O)]where pyOAc is 2-pyridylacetate).²³ More recent work led to there-examination of whether the chelating function is necessary becausethe acetate group appears to not only enhance aqueous solubility butalso slow down the rate of hydrolysis in these water-soluble TPAderivatives.²⁴

Inventive Example 5

This present invention provides new, representative TPA compoundscontaining the acetate ligands as leaving groups in the trans axis. Useof acetate ligands (and carboxylate ligands in general) results inaqueous solubility and hydrolysis rates which leads to more desirablebehavior in vivo. In addition, the biological profiles for these transacetates indicate a lack of cross-resistance (collateral sensitivity) intumor cells resistant to either cisplatin or oxaliplatin. TheTPA-acetate derivatives are significantly more cytotoxic in manycis-DDP-resistant cell-lines than in the parent cis-DDP-sensitivecell-lines, an encouraging and remarkable finding. This new series ofcompounds is the first example of an N₂O₂ donor set for cytotoxictransplatinum compounds. Further, the “trans-carboxylate” strategy is ageneral one, applicable to all donor ligands such as alicyclic amines,iminoethers and heterocyclic aliphatic amines such as piperazine andpiperidine.

The general synthetic pathway is described in Example 6. The diiodocompounds are especially useful intermediates as they are precursors ofnitrato compounds which were isolated in all cases for use as controlsin chemical studies. ^(25,26.)Further, the acetone solubility of thediiodo compounds precludes the need for use of high-boiling solventssuch as DMF which are necessary to solubilize the sparingly solublechlorides. Characterization of all representative compounds was byelemental analysis, UV/Vis and NMR spectroscopy. Purity was assessed byHPLC.²⁷ An X-ray crystal structure determination oftrans-[Pt(OAc)₂(py)₂] also confirmed the proposed structure.

The aqueous solubility of the acetate compounds and the aciddissociation constants of the corresponding aqua species are given inTable 1. Use of acetates enhances aqueous solubility which is dependenton the exact nature of the donor ligands. The pK_(a) values weredetermined by potentiometric titration of solutions oftrans-[Pt(H₂O)₂(L)(L′)]²⁺ (formed by the dissolution of the isolatedtrans-[Pt(NO₃)₂(L)(L′)]) with NaOH. The first pK_(a) is significantlylower than that found for cisplatin (pK_(a1)=5.37).²⁸ There is littlevariation amongst the various planar ligands, a feature also noted inthe series trans-[PtCl₂(NH₃)(X-pyr)] (X=2,3,4-Me).³⁰

TABLE 1

Physical Properties of TPA-(Acetate) and pK_(a) Values of theCorresponding TPA Diaqua Complexes at Ambient Temperature^(a) ComplexSolubility, mg/mL pK_(a1) pK_(a2) trans-[Pt(OAc)₂(NH₃)₂] 21 4.0^(b) 7.08trans-[Pt(OAc)₂(py)₂] 15 3.87 6.7 trans-[Pt(OAc)₂(NH₃)(quin)] 1.6 3.897.01 trans-[Pt(OAc)₂(NH₃)(iquin)] 5.2 3.78 6.92 ^(a)pH titration curveswere obtained from the potentiometric titrations of 10⁻³ M solutions oftrans-[Pt(OH₂)₂(L)(L′)] (produced from the corresponding dinitratocompounds) by a standardized NaOH solution (9.5 × 10⁻⁴ M). Solubilitywas measured by sequential addition of water, agitation for 15 minutesuntil solution was no longer clear. ^(b)Literature values 4.35 and 7.40by NMR.²⁹

For hydrolysis studies, 10⁻³ mM complex was dissolved in 1 mL ofnanopure water. The pH values of the samples were controlled in regularintervals and readjusted if necessary by addition of HNO₃ (1 M, 0.1 M,0.01 M) or NaOH (1 M, 0.1 M, 0.01 M). Aliquots of 20 μL were taken fromthe bulk solution for HPLC analysis. The overall scheme may be describedas follows:

The aquated species were identified by comparison with the hydrolysisprofile produced by the corresponding (labile) nitrate compounds. Therate constants for the first aquation step k1 are given in Table 2 andcompared with other platinum complexes containing one or two labileligands. The rate for the TPA acetates (approx. 10⁻⁷ s⁻¹) issignificantly slower than those of Pt complexes containing chloroligands (10⁻⁵ s⁻¹). In particular, these rates are 2-3 orders ofmagnitude lower than that of transplatin itself. The rate of aquationtends toward that of carboplatin which in phosphate buffer at 310 K gavea hydrolysis rate of 7.2×10⁻⁷ s⁻¹ (but in aqueous solution gives abarely measurable rate constant of <5×10⁻⁹ s⁻¹, which is modulated bythe presence of buffer, nucleophile or acid). ^(31,32)No evidence forformation of Pt(TV) species during hydrolysis is detected under theseconditions.³³

TABLE 2 Comparison of Hydrolysis Rates of TPA Acetates and OtherPlatinum Compounds. Compound k₁ s⁻¹ trans-[Pt(OAc)₂(py)₂] (3.2 ± 0.3) ×10⁻⁷ trans-[Pt(OAc)₂(NH₃)(quin)] (3.7 ± 0.1) × 10⁻⁷trans-[Pt(OAc)₂(NH₃)(iquin)] (7.4 ± 1.5) × 10⁻⁷ Cisplatin³⁴ 5.18 × 10⁻⁵Carboplatin^(31,32) <10⁻⁸ Transplatin³⁵ 19 × 10⁻⁵ [PtCl(dien)]+,³⁶ (6.50± 0.13) × 10⁻⁵The acetate group (and carboxylate group in general) is a weak ligandand has a low trans influence and trans effect. The lability ofmonofunctional carboxylate is greater than chloride. Incis-[Pt(amine)₂(RCO₂)₂], as exemplified by cis-[Pt(PrNH₂)₂(ClCH₂CO₂)₂],analysis of the kinetics of substitution by water of the firstcarboxylate ligand (rate constant=4.38×10⁻⁵ s⁻¹) suggested a strong cislabilising effect of the mutually cis carboxylates.³⁷ This effect isabsent in [Pt(dien)(RCO₂)]⁺.³⁸ These measured rate constants aresignificantly higher than that measured here for the trans geometry.Placing two weak carboxylate ligands in a mutually trans-axial positionof a square-planar Pt(II) compound results in little driving force forligand substitution. Thus, surprisingly, trans-carboxylates aresignificantly more inert than simple considerations of themonofunctional carboxylate would indicate.

The IC₅₀ values in an ovarian cancer cell line panel are given in Table3. It is noteworthy that the acetate compounds maintain cytotoxicitywith low resistance factors in all cases. The trans-[Pt(OAc)₂(NH₃)₂]compound is also very water-soluble but the cytotoxicity of >100 μMconfirms the requirement for a planar amine to enhance cytotoxicity inthe trans geometry.

TABLE 3 Evaluation of TPA-Acetate Derivatives in Human Ovarian CancerCell Line Panel (96 h exposure)^(a) Compound A2780 RF CH1 RF 41M RFcis-[PtCl₂(NH₃)₂] 0.1 1.4 (6.7) (6.1) trans-[Pt(OAc)₂(NH₃)(quin)] 13.017.0 22.0 (1.46) (0.48) (1.00) trans-[Pt(OAc)₂(NH₃)(iquin)] 13.0 20.022.0 (1.69) (0.37) (0.26) trans-[Pt(OAc)₂(py)₂] 12.8 19.0 14.0 (0.90)(0.22) (0.32) ^(a)IC₅₀ (concentration necessary to inhibit growth at50%) in μM. Values in parentheses are resistant factors, [RF = (IC₅₀Resistant)/(IC₅₀ Sensitive)]. Assays performed as per ref.¹⁶.

A study of the cytotoxicity of the trans-[PtCl₂(L)(L′)] series acrossthe NCI human tumor cell line panel showed a unique profile and activityin both cisplatin and oxaliplatin-resistant cells.³⁹ This property ismaintained for the new acetate compounds and, indeed, resistance factorsappear to be even lower than for the parent chlorides, as clearly seenin comparison of a representative compound in FIG. 1. Thus, the newcompounds retain the desirable features of the parent compounds and theprofile of cytotoxicity is similar.³⁹

The chemistry of the new TPA acetate compounds described here is likelyto affect all the principal pharmacological factors affecting platinumtoxicity—cellular uptake, structure and nature of DNA lesions and theextent of deactivating metabolic interactions. In agreement, besides theexpected profile of DNA binding,⁸ cellular uptake is greatly enhancedfor acetate compounds.⁴⁰ This combination of modulation of chemicalproperties may be reasonably expected to produce the cytotoxicityprofile distinctly different from that of cisplatin and its congeners.

The compounds described here are the first transplatinum compoundscontaining an N₂O₂ donor set, similar to carboplatin and oxaliplatin.The reactivity of the new series suggests they are best considered as“carboplatin” analogs but in the trans geometry. The reactivity andaquation rates can be further modulated by variation of carboxylateligand.⁴⁰ Carboplatin is significantly less potent than cisplatin incell culture and is safely administered clinically at higher dosesbecause of diminished side-effects. Application of this strategy to thediverse set of cytotoxic transplatinum compounds described in theliterature could lead to the selection of clinical candidates with aprofile genuinely different to that of the currently used agents.

References for Example 5

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p0 4. Wong, E.; Giandomenico, C. M. Current Status of Platinum-BasedAntitumor Drugs. Chem. Rev. 1999, 99, 2451-2466.

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Platinum complexes with imino ethers or cyclic ligands mimicking    imino ethers: synthesis, in vitro antitumour activity, and DNA    interaction properties. J. Biol. Inorg. Chem. 2004, 9, 768-780.-   10. Coste, F.; Malinge, J. M.; Serre, L.; Shepard, W.; Roth, M.;    Leng, M.; Zelwer, C. Crystal structure of a double-stranded DNA    containing a cisplatin interstrand cross-link at 1.63 A resolution:    hydration at the platinated site. Nucleic Acids Res. 1999, 27,    1837-1846.-   11. Huang, H.; Zhu, L.; Reid, B. R.; Drobny, G. P.; Hopkins, P. B.    Solution structure of a cisplatin-induced DNA interstrand    cross-link. Science 1995, 270, 1842-1845.-   12. Brabec, V.; Leng, M. DNA interstrand cross-links of    trans-diaminedichloroplatinum(II) are preferentially formed between    guanine and complementary cytosine residues. Proc. Natl. Acad. Sci.    U.S.A. 1993, 90, 5345-5349-   13. Kartalou, M.; Essigmann, J. M. Recognition of cisplatin adducts    by cellular proteins. Mutat. Res. 2001, 478, 1-21.-   14. Jamieson, E. R.; Lippard, S. J. Structure, recognition, and    processing of cisplatin-DNA adducts. Chem. Revs, 1999, 99,    2467-2498.-   15. van Beusichem, M.; Farrell, N. Activation of the trans geometry    in platinum antitumor complexes. Synthesis, characterization, and    biological activity of complexes with the planar ligands pyridine,    N-methylimidazole, thiazole, and quinoline. Crystal and molecular    structure of trans-dichlorobis(thiazole)platinum(II). Inorg. Chem.    1992, 31, 634-639.-   16. Farrell, N.; Kelland, L. R.; Roberts, J. D.; van Beusichem, M.    Activation of the trans geometry in platinum antitumor complexes: a    survey of the cytotoxicity of trans complexes containing planar    ligands in murine L1210 and human tumor panels and studies on their    mechanism of action. Cancer Res., 1992, 52, 5056-5072.-   17. Mellish, K. J.; Barnard, C. F. J.; Murrer, B. A.; Kelland, L. R.    DNA-binding properties of novel cis- and trans-platinum-based    anticancer agents in 2 human ovarian carcinoma cell lines. Int. J.    Cancer, 1995, 62, 717-723.-   18. Montero, E. I.; Diaz, S.; Gonzalez-Vadillo, A. M.; Perez, J. M.;    Alonso, C.; Navarro-Ranninger, C. Preparation and Characterization    of Novel trans-[PtCl2(amine)(isopropylamine)] Compounds: Cytotoxic    Activity and Apoptosis Induction in ras-Transformed Cells. J. Med.    Chem. 1999, 42, 4264-4268.-   19. Khazanov E.; Barenholz Y.; Gibson D.; Najajreh Y. Novel    apoptosis-inducing trans-platinum piperidine derivatives: synthesis    and biological characterization. J. Med. Chem. 2002, 45, 5196-204.-   20. Najajreh Y.; Perez J. M.; Navarro-Ranninger, C.; Gibson, D.    Novel soluble cationic trans-diaminedichloroplatinum(II) complexes    that are active against cisplatin resistant ovarian cancer cell    lines. J. Med. Chem. 2002, 45, 5189-95.-   21. Brabec, V. DNA modifications by antitumor platinum and ruthenium    compounds: their recognition and repair. Prog. Nucleic Acid Res.    Mol. Biol. 2002, 71, 1-68-   22. Leng M.; Locker D.; Giraud-Panis M. J.; Schwartz A.; Intini F.    P.; Natile, G.; Pisano, C.; Boccarelli A.; Giordano, D.;    Coluccia, M. Replacement of an NH(3) by an iminoether in transplatin    makes an antitumor drug from an inactive compound. Mol. Pharmacol.    2000, 58, 1525-35.-   23. Bierbach, U.; Sabat, M.; Farrell, N. Synthesis, crystal    structure, and cytotoxicity of trans-[Pt(PyAc-N,O)Cl(NH3)]:    Unprecedented inversion of the cis-geometry requirement for    platinum-based antitumor complexes. Inorg. Chem. 2000, 39,    1882-1890.-   24. Quintal, S. M. O.; Qu, Y.; Gomez-Quiroga, A.; Moniodis, J.;    Nogueira, H. I. S.; Farrell, N. Pyridine-carboxylate complexes of    platinum. Effect of N,O-chelate formation on model bifunctional    DNA-DNA and DNA-protein interactions. Inorg. Chem. 2005, 44,    5247-5253.-   25. Bierbach, U.; Farrell, N. Structural and reactivity studies on    the ternary system guanine/methionine/trans-[PtCl2(NH3)L] (L=NH3,    quinoline): implications for the mechanism of action of nonclassical    trans-platinum antitumor complexes. J. Biol. Inorg. Chem. 1998, 3,    570-580.-   26. Souchard, J. P.; Wimmer, F. L.; Ha, T. T. B.; Johnson, N. P. A    rapid method for the synthesis of water-soluble platinum(II) amine    and pyridine complexes. J. Chem. Soc., Dalton Trans. 1990, 307-310.-   27. trans-[Pt(OAc)2(NH3)(quin)] 1H NMR in D2O: 9.71 (d, 1 H), 9.24    (d, 1 H), 8.48 (d, 1 H), 8.10 (m, 1 H), 7.75 (m, 1 H), 7.57 (m, 1    H), 1.80 (s, 6 H). Anal. Calcd. (Found) for C13H16N2O4Pt: % C, 33.99    (33.97); % H, 3.51 (3.12); % N, 6.10 (6.02); % Cl, 0.00 (0.21).-   Purity by HPLC: >99.9% pure (H2O, MeOH mobile phase).-   Trans-[Pt(OAc)2(NH3)(iquin)] 1H NMR in CD3OD: d 9.20 to 7.63, iquin    region; 4.38 (br), NH3; 1.85 (s), 2(02CCH3). 1H NMR in D2O: d 9.12    to 7.76 iquin region; 2.00 (s), 2(O2CCH3). Anal. Calcd. (Found) for    C13H16N2O4Pt.(H2O): % C, 32.71 (32.44); % H, 3.80 (3.41); % N, 5.87    (5.75); % Cl, 0.00 (0.12). Purity by HPLC: 100% pure (H2O, MeOH    mobile phase).-   trans-[Pt(OAc)2(NH3)2] Anal. Calcd. (Found) for C4H12N2O4Pt: % C,    13.84 (13.93); % H, 3.48 (2.94); % N, 8.07 (8.18); Cl, 0.00 (<0.10).-   trans-[Pt(OAc)2(py)2] Anal. Calcd. for C14H16N204Pt: % C, 35.67; %    H, 3.42; % N, 5.94.-   Found: % C, 35.67; % H, 3.18; % N, 5.78. 1H NMR: d.1H NMR in CD3OD:    (CD3OD): d 8.67 (d, 2 H, 2 H2,6), 7.98 (t, 1 H, H4), 7.51 (t, 2 H, 2    H3,5), 1.88 (s, 6 H, O2CCH3). Purity by HPLC: 99.4% (gradient    elution profile H2O/CH3CN 15:85).-   28. Bemers-Price, S. J.; Frenkiel, T. A.; Frey, U.; Ranford, J. D.;    Sadler, P. J. J. Chem. Soc., Chem. Comm. 1992, 789-791.-   29. Appleton, T. G.; Bailey, A. J.; Bamlham, K. J.; Hall, J. R.    Aspects of the Solution Chemistry of trans-Diammineplatinum(II)    Complexes. Inorg. Chem. 1992, 31, 3077-3082.-   30. McGowan, G., Parsons, S.; Sadler, P. J. The contrasting    chemistry of cis and trans PtII diamine anticancer compounds:    hydrolysis studies of picoline complexes.-   31. Canovese, L.; Cattalini, L.; Chessa, G.; Tobe, M. L., Kinetics    of the displacement of cyclobutane-1,1-dicarboxylate from    diammine(cyclobutane-1,1-dicarboxylato)platinum(II) in aqueous    solution. J. Chem Soc., Dalton Trans. 1988, 2135-2140.-   32. Frey, U.; Ranford, J. D.; Sadler, P. J. Ring-opening reactions    of the anticancer drug carboplatin: NMR characterization of    cis-[Pt(NH3)2(CBDCA-O)(5′-GMP-N7)] in solution. Inorg. Chem. 1993,    32, 1333-1340.-   33. Pizarro, A. M.; Munk, V. P.; Navarro-Ranninger, C.; Sadler, P.    J., Hydrolysis triggers oxidation of a trans Diamine Platinum(II)    Anticancer Complex. Angew. Chem. Int. Ed., 2003, 42, 5339-5342.-   34. Miller, S. E.; House, D. A. The hydrolysis products of    cis-diammine-dichloroplatinum(II). 2. The kinetics and formation and    anation of the cis-diamminedi(aqua)platinum(II) cation. Inorg. Chim.    Acta 1989, 166, 189-197.-   35. Segal, E.; Le Pecq, J. B. Role of ligand exchange processes in    the reaction kinetics of the antitumor drug    cis-diaminedichloroplatinum(II) with its targets. Cancer Res. 1985,    45, 492-497.-   36. Marti, N.; Hoa, G. H.; Kozelka, J. Reversible hydrolysis of    [PtCl(dien)]+ and [PtCl(NH3)3]+. Determination of the rate constants    using UV spectrophotometry. Inorg. Chem. Commun. 1988, 1, 439-.445-   37. Canovese, L.; Cattalini, L.; Chessa, G.; Tobe, M. L.; Kinetics    of the displacement of cyclobutane-1,1-dicarboxylate from    diammine(cyclobutane-1,1-dicarboxylato)platinum(II) in aqueous    solution. J. Chem. Soc., Dalton Trans. 1988, 2135-2140.-   38. Canovese, L.; Cattalani, L.; Gemelli, L.; Tobe, M. L. Acid- and    base-catalyzed displacement of the carboxylate ligand from    [Pt(dien)(RCO2)]+(dien=1,5-diamino-3-azapentane; R═CH2Cl, CHCl2, or    CCl3) in aqueous solution. J. Chem. Soc., Dalton Trans. 1988,    1049-1052.-   39. Murphy, R. F.; Farrell, N.; Aguila, A.; Okada, M.; Balis, F. M.;    Fojo, T. Accumulation of novel transplatinum complexes in cisplatin    and oxaliplatin resistant cell lines overcomes resistance. Proc.    AACR, 2005, 4109.-   40. Fojo, T.; Farrell, N.; Orthuzar, W.; Tanimura, H.; Stein, J.;    Myers, T. G. Identification of non-cross-resistant platinum    compounds with novel cytotoxicity profiles using the NCI anticancer    drug screen and clustered image map visualizations. Crit. Rev.    Hematol. Oncol. 2005, 53, 25-34.

Example 6 Effect of Carboxylate Leaving Groups and Steric Hindrance onChemical and Biological Properties of Trans-Platinum Planar AmineCompounds

Replacement of NH₃ by a planar amine L to give trans-[PtCl₂(L)(L′)](L=NH₃, L′=pyridine or substituted pyridine, quinoline, isoquinoline,thiazole; L=L′=pyridine, thiazole), greatly enhances the cytotoxicity ofthe transplatinum geometry. The “parent” compound trans-[PtCl₂(NH₃)₂] istherapeutically inactive. Modification of the ligands to an [N₂O₂] donorset, where O represents an acetate leaving group, enhances aqueoussolubility whilst retaining the cytotoxicity of the parent chloridecompounds. The effect of two mutual trans leaving groups but with weaktrans influence is to impart remarkable chemical stability on thestructure. This strategy is analogous to the use of the inertdicarboxylate leaving groups in the clinical compounds carboplatin andoxaliplatin. In this paper, systematic modification of the stericeffects of carrier pyridine groups and, especially, carboxylate leavinggroups in trans-[Pt(O₂CR)₂(NH₃)(pyr)] is shown to modulate aqueoussolubility and hydrolysis to the activated aqua species. The resultspresented here demonstrate the utility of the “carboxylate strategy” in“fine-tuning” the chemical and pharmacokinetic properties in the designof clinically relevant transplatinum complexes.

Platinum complexes in the trans geometry are of interest for theirbiological properties. Substitution of NH₃ in trans-[PtCl₂(L)(L′)] givescomplexes with cytotoxicity in the micromolar range. Since the firstpublication of this phenomenon using planar amines 1-3 (pyridine,thiazole, quinoline, isoquinoline etc.) a range of amine ligands havebeen employed, including iminoethers, alicyclic amines and heterocyclicaliphatic amines.⁴⁻⁸ In general, these complexes exhibit enhancedcytotoxicity with respect to the parent transplatin and are usuallynon-cross-resistant with cisplatin. Complexes containing Trans-PlanarAmines (TPA compounds) exhibit a unique cytotoxicity profile in theNational Cancer Institute (NCI) tumor panel and induce topoisomeraseI-DNA complexes in human tumor cells.⁹⁻¹⁰ To address the poor aqueoussolubility and relatively high chemical reactivity of thetrans-[PtCl₂(L)(L′)] structure we have introduced the use ofcarboxylates as leaving groups in the first examples of cytotoxictransplatinum complexes containing [N₂O₂] ligand donor sets.¹¹ Complexessuch as. trans-[PtOAc)²(pyr)²] are very water-soluble and surprisinglystable toward hydrolysis—resembling carboplatin in their reactivity. Asa series, the complexes are cytotoxic in both cisplatin andoxaliplatin-resistant cells and show remarkably high cellularuptake.^(11,12)

In Example 5 we suggested that the “carboxylate strategy” could beextended to all classes of transplatinum complexes. A report on somesimple examples, using compounds containing the alicyclic amine motif,¹³analogs of compounds first developed by Navarro-Raninger et al.,⁷ hassince confirmed this suggestion.

The pharmacological properties of trans-[Pt(O₂CR)₂(L)(L′)] can inprinciple be modified by steric and electronic effects of the donorgroups as well as in the leaving carboxylate ligands. In this example,we report on the synthesis and characterization of a structurallysimilar set, trans-[Pt(O₂CR)₂(NH₃)(L)], and show that systematicvariation of donor ligand (L=pyridine or substituted pyridine, 2-pic,3-pic, or 4-pic) and R allows for a range of aquation rates and alsocytotoxicity. Thus the general [PtN₂O₂] structure is capable of“fine-tuning” to enhance biological activity.

The platinum complexes from this study are presented in FIG. 2. Thesynthesis and characterization of the intermediates and complexes1a-c,¹¹ 2a, 3a & 4a,¹⁴ 3b-c & 4b-c¹² have been previously reported. Thesyntheses of complexes 2b, 2c,¹⁶ 4d,¹⁷ 4e,¹⁸ 4f,¹⁹ 4g²⁰ and 4h wereadapted from literature procedures,¹¹ and purity was confirmed byHPLC.²² The ¹H NMR chemical shifts and elemental analysis of thesecompounds is reported in the supplementary information. The silver saltsof the carboxylates were prepared as described below.¹⁵ The pathway tothe final carboxylate complex takes advantage of the increased transinfluence of the halides over the N-donor ligands. In all examples,synthesis from the chloride complex to the iodide intermediate wasquantitative in yield, and yields of the final carboxylate complexeswere much higher (50-70%) compared to recently published methods.¹³

TABLE 1 Solubility of TPA Carboxylates and pKa of the Corresponding TPADiaqua Complexes at Ambient Temperature. TPA Substituted TPA AcetatesCarboxylates Solubility Solubility pK_(a1) pK_(a2) (mg/mL) (mg/mL) 1c¹¹3.73 6.8 11.6 4d 22 2c 4.03 7.1 10 4 35 3c^(11,14) 3.97 6.78 11.4 4f0.29 4c^(11,14) 3.94 6.88 13.2 4g Insoluble 4h Insoluble

The solubility of the complex series was measured at 37° C. and ispresented in Table 1. Replacement of the halogens with acetate ligandssignificantly enhanced the aqueous solubility of TPAs as previouslyreported.¹¹ Aqueous solubility of the acetate series is influenced bythe steric hindrance of the heterocycle (pyridine, quinoline etc.) ¹¹but there is little difference seen for the various substitutionpatterns around the pyridine ring. In contrast, significant differencesare noted upon changing the carboxylate group. For the series of 4-piccomplexes, increase in hydrogen bonding capability leads to enhancedsolubility as seen for the formate 4e and hydroxyacetate 4d derivatives,while the chloroacetate 4f, trifluoroacetate 4g and benzoate 4hderivatives show reduced solubility.

The pKa of 1c was determined from the potentiometric titration ofsolutions of trans-[Pt(H₂O)₂(L)(L′)]²⁺ as per Ma.¹¹ This is presentedTable 1 in comparison to the pKa values of the other compounds of theseries 2-4c. The result is consistent with previous data^(11,14) andconfirms the effect on pKal of substitution of an ammine NH₃ or diamine(ethylenediamine, 1,2-diaminocyclohexane) by a π-acceptorligand.^(14,24)

The initial hydrolysis of selected TPA carboxylates (2-4c, 4d, 4e) wasmonitored by HPLC over a period of 12 h at 37° C., and initialhydrolysis rate constants were calculated using the program SCIENTIST(Version 2.0, MicroMath, Inc.) and are presented in FIG. 3. The slowestinitial hydrolysis rate was found for complex 2ct-[Pt(OAc)₂(NH₃)(2-pic)], with just 4% hydrolysis occurring after 9 h.The hydrolysis of the formate complex 4f was the fastest, with 24%hydrolysis occurring after 9 h. The hydrolysis rates of the acetatecomplexes followed the pattern 2c<4c<3c. These results may be contrastedwith those for the analogous dichloride complexes where the firsthydrolysis step for formation of the monoaqua complex is relatively fastwith k₁=2.6, 12.7 and 5.2×10⁻⁵ s⁻¹ (I=0.1 M) for 2-pic, 3-pic and 4-picrespectively.¹⁴ In contrast, the rates observed for the analogousacetate derivatives were k₁=1.07, 3.52 and 3.26×10⁻⁶ s⁻¹ (I=0.02 M) for2c, 3c and 4c respectively. Thus, hydrolysis of the acetate complexes isslowed by an approximate order of magnitude compared to their directchloro analogs but there is little difference in the present casebetween 3- and 4-picoline. Steric hindrance therefore predominates overthe electronic effects of the methyl group. In the case of the chloridecomplexes, the 3-pic directs less electron density to the platinumrendering it less nucleophilic relative to the 2-pic and 4-pic. 14 Weobserve the same trend in the acetate complexes, but the effect islessened due to the carboxylate group being a weak ligand, with a lowtrans influence and trans effect.¹¹

The hydrolysis rates of the carboxylate complexes followed the samepattern as the solubility, with 4c<4d<4e and values of k₁=3.26, 5.61 and10.7×10⁻⁶ s⁻¹ (I=0.02 M) respectively. The measured rate of 10.7×10⁻⁶s⁻¹ for the formate complex 4e is still slower than that of thechloro-analog and even that of cisplatin (51.8×10⁻⁶ s⁻¹ at 20° C.).²⁵

The slow hydrolysis is a unique feature of two carboxylate ligands in atrans-axial position and can be modulated by choice of carboxylate. Toexamine how this might affect possible reactions with biomolecules, apreliminary study of the reactions of 4e with guanosine-5′-monophosphate(5′-GMP, model for DNA) and N-acetyl-L-methionine (N-AcMet, model forsulfur ligand metabolism) was made by ¹⁹⁵Pt NMR Spectroscopy. In thecase of 5′-GMP the peak of the starting material at −1450 ppm is onlygradually replaced by a new peak at −2365 ppm. This shift is consistentwith the formation of a species that has a PtN₄ coordination sphere,²⁶due to replacement of both formate groups. Approximately 50% reaction(as judged by the intensity of the ¹H H8 signal and ¹⁹⁵Pt NMR signals),occurred within 24h. For N-AcMet the starting material was approximately50% of total intensity even after 8 h. A new species was observed withδ(¹⁹⁵Pt) at −1775 ppm, which formed after one hour, and did not exceed15% of the total reaction product over 10 h. The chemical shift isconsistent with a PtN₂O₂ coordination sphere—this could arise fromdisplacement of formate by H₂O/OH or an O-bound methionine.²⁷ Thisspecies preceded the formation of the N-AcMet product at −3320 ppmconsistent with a PtN₂S₂ coordination sphere,trans-[Pt(NH₃)(4-pic)(NAcMet)₂],^(23,28) which was 40% of the totalplatinum species after 10 h. This reaction can be contrasted to that oftrans-[PtCl₂(NH₃)₂], whose reaction with N-AcMet is complete within 5h.²⁸ These results emphasize the previously-made analogy between thetransplatinum [N₂O₂] ligand donor set and carboplatin, and show thatsubstitution reactions with potentially deactivating biomolecules may besignificantly retarded in comparison with chloro analogs. Whether thepossible formation of an O-bound methionine species is a reflection ofthe low pKal and the electronic effects of a π-acceptor ligand is worthyof further investigation.

The MTT assay was used to determine growth inhibition of the platinumdrugs in the human ovarian cancer cell line A2780.²⁹ All TPAcarboxylates tested exhibit cytotoxic behavior in the micromolar range.The most cytotoxic compound, 4e, trans-[Pt(OFm)₂(NH₃)(4-pic)], was alsothat which displayed the fastest hydrolysis, as well as being mostsoluble, FIG. 4. The cytotoxicity of the carboxylate compounds increasesin the order 4c<4d<4e (OAc<OAcOH<OFm), suggesting that the nature of thecarboxylate leaving group is related to cytotoxicity. The cytotoxicityof the acetate compounds increases in the order 2c<3c<4c(2-pic<3-pic<4-pic), suggesting that the steric hindrance of the methylgroup can influence cytotoxicity, with the more sterically-hindered2-pic complex being the most cytotoxic of the acetate compounds.

It is remarkable that the complexes with carboxylate leaving groupsdisplay, in general, cytotoxicity equivalent to the parent chlorides,despite their differences in reactivity.^(1,2,12,14) In contrast,carboplatin is significantly less cytotoxic than cisplatin on a molarbasis. Previous studies have shown significantly enhanced cellularuptake of the acetate derivatives^(11,12) and it is reasonable tosuggest that these results may reflect the overall modulation of thepharmacological properties responsible for cellular toxicity—uptake,nature and frequency of DNA adducts and the extent of metabolizing“deactivating” reactions. Thus, the results presented here demonstrateagain the utility of the “carboxylate strategy” in the design ofclinically relevant transplatinum complexes.

In summary, replacing an ammine in inactive trans-platin complexes witha planar amine greatly enhances the cytotoxicity, such as intrans-[PtCl₂(NH₃)(L′)] (where L′=py, pic, iquin). Systematicmodification of the steric effects of carrier pyridine groups andcarboxylate leaving groups in trans-[Pt(O₂CR)₂(NH₃)(pyr)] is shown tomodulate aqueous solubility and hydrolysis to the activated aquaspecies, while retaining cytotoxicity. Especially, the nature of thecarboxylate ligand affects both hydrolysis (production of thepharmaceutically active aquated species) and cytotoxicity. Thus the“carboxylate strategy” is especially useful in “fine-tuning” thechemical and pharmacokinetic properties in the design of clinicallyrelevant transplatinum complexes.

References for Example 7

-   (1) Van Beusichem, M.; Farrell, N. Inorg. Chem. 1992, 31, 634-639.-   (2) Farrell, N.; Kelland, L. R.; Roberts, J. D.; Van Beusichem, M.    Cancer Res. 1992, 52, 5065-5072.-   (3) Farrell, N. Cancer Invest. 1993, 11, 578-589.-   (4) Coluccia, M.; Nassi, A.; Loseto, F.; Boccarelli, A.;    Mariggio, M. A.; Giordano, D.; Intini, F. P.; Caputo, P.;    Natile, G. J. Med. Chem. 1993, 36, 510-512.-   (5) Khazanov, E.; Barenholz, Y.; Gibson, D.; Najajreh, Y. J. Med.    Chem. 2002, 45, 5196-5204.-   (6) Najajreh, Y.; Perez, J. M.; Navarro-Ranninger, C.; Gibson, D. J.    Med. Chem. 2002, 45, 5189-5195.-   (7) Montero, E. I.; Diaz, S.; Gonzalez-Vadillo, A. M.; Perez, J. M.;    Alonso, C.; Navarro-Ranninger, C. J. Med. Chem. 1999, 42, 4264-4268.-   (8) Natile, G.; Coluccia, M. Coord. Chem. Rev. 2001, 216-217,    383-410.-   (9) Fojo, T.; Farrell, N.; Ortuzar, W.; Tanimura, H.; Weinstein, J.;    Myers Timothy, G. I Crit. Rev. Oncol./ Hematol. 2005, 53, 25-34.-   (10) Murphy, R. F.; Farrell, N.; Aguila, A.; Okada, M.; Balis, F.    M.; Fojo, T. Proc. Am. Assoc. Cancer Res. 2005, 46, [Abstract    #4109].-   (11) Ma, E. S. F.; Bates, W. D.; Edmunds, A.; Kelland, L. R.; Fojo,    T.; Farrell, N. J. Med. Chem. 2005, 48, 5651-5654.-   (12) Quiroga, A. G.; Perez, J. M.; Alonso, C.; Navarro-Ranninger,    C.; Farrell, N. J. Med. Chem. 2006, 49, 224-231.-   (13) van Zutphen, S.; Pantoja, E.; Soriano, R.; Soro, C.; Tooke, D.    M.; Spek, A. L.; den Dulk, H.; Brouwer, J.; Reedijk, J. Dalton    Trans. 2006, 1020-1023.-   (14) McGowan, G.; Parsons, S.; Sadler, P. J. Inorg. Chem. 2005, 44,    7459-7467.-   (15) AgOAcOH (AgO₂CCH₂OH, OAcOH=hydroxyacetate) was prepared by    adding hydroxyacetic acid (0.0526 mol) to a suspension of Ag₂O (4.32    mmol) in 150 ml H₂O. The mixture was stirred for 2 h in the dark,    filtered through Celite and reduced until a white crystalline solid    formed. Yield: 1.40 g (89%). AgOAcCl (AgO₂CCH₂Cl,    OAcCl=chloroacetate), AgOFm (AgO₂CH, OFm=formate), AgTfa (AgO₂CCF₃,    Tfa=trifluoroacetate) and AgOBz (AgO₂CC₆H₅, OBz=benzoate) were    prepared similarly.-   (16) trans-[Pt(OAc)₂(NH₃)(2-pic)] 2c. ¹⁹⁵Pt NMR (d₆-acetone) δ    (ppm): −1420. IR (cm-1) 1633 m (C═O).-   (17) trans-[Pt(OAcOH)₂(NH₃)(4-pic)] 4d, ¹⁹⁵Pt NMR (D₂O) δ (ppm):    −1454. IR (cm-1) 1645 s (C═O).-   (18) trans-[Pt(OFm)₂(NH₃)(4-pic)] 4e, ¹⁹⁵Pt NMR (D₂O) δ (ppm):    −1450. IR (cm-1) 1614 s (C═O).-   (19) trans-[Pt(OAcCl)₂(NH₃)(4-pic)] 4f, ¹⁹⁵Pt NMR (CD₃OD) δ (ppm):    −1403. IR (cm-1) 1619 s, 1645 m, 1668 m (C═O).-   (20) trans-[Pt(Tfa)₂(NH₃)(4-pic)] 4g, ¹⁹⁵Pt NMR (CD₃OD) δ (ppm):    −1402. IR (cm-1) 1680, 1705 d, s(C═O).-   (21) trans-[Pt(OBz)₂(NH₃)(4-pic)] 4h, ¹⁹⁵Pt NMR (CD₃OD) δ (ppm):    −1420, IR (cm-1) 1633 m, 1620 m (C═O).-   (22) RP HPLC, C18 Hydrosphere column (Phenomenex),    Water/Acetonitrile gradient-   (23) Norman, R. E.; Ranford, J. D.; Sadler, P. J. Inorg. Chem. 1992,    31, 877-888.-   (24) Summa, N.; Schiessl, W.; Puchta, R.; van Eikema Hommes, N.; van    Eldik, R. Inorg. Chem. 2006, 45, 2948-2959.-   (25) Miller, S. E.; House, D. A. Inorg. Chim. Acta. 1989, 166,    189-197.-   (26) Fontes, A. P. S.; Oskarsson, A.; Loevqvist, K.; Farrell, N.    Inorg. Chem. 2001, 40, 1745-1750.-   (27) Barnham, K. J.; Frey, U.; Murdoch, P. d. S.; Ranford, J. D.;    Sadler, P. J.; Newell, D. R. J. Am. Chem. Soc. 1994, 116,    11175-11176.-   (28) Oehlsen, M. E.; Hegmans, A.; Qu, Y.; Farrell, N. J. Biol.    Inorg. Chem. 2005, 10, 433-442.-   (29) 6500 cells per well, 37° C./5% CO₂, 72 h. IC₅₀ values were    determined graphically

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

1. A trans-platinum (II) complex of formulatrans-[Pt(carboxylato)₂(L)(L′)] where carboxylato =a ligand derived froman anion of a carboxylic acid, and wherein said trans-platinum (II)complex is represented by formula (I) as follows:

wherein i) RCOO— is a carboxylate anion of a carboxylic acid and R is Hor an alkyl group which may be substituted or unsubstituted; and ii) Land L′ are both planar heterocyclic amines, or one of L or L′ is aplanar heterocyclic amine and the other is selected from the groupconsisting of NH₃ a branched aliphatic amine, an iminoether, a primaryamine, a secondary amine and an aliphatic nitrogen-containingheterocycle.
 2. The trans-platinum (II) complex of claim 1, wherein L isa planar amine and L′ is NH₃.
 3. The trans-platinum (II) complex ofclaim 1, wherein the aliphatic nitrogen-containing heterocycle isselected from the group consisting of piperazine, piperidine andpyrazole.
 4. The trans-platinum(II) complex of claim 1, wherein saidcarboxylato ligand is selected from the group consisting of formate,acetate, hydroxyacetate, chloroacetate, trifluoroacetate and lactateincluding optical isomers thereof.
 5. The trans-platinum (II) complex ofclaim 1, wherein said planar heterocyclic amine is selected from thegroup consisting of pyridine, substituted pyridines, quinoline,subsituted quinolines, isoquinoline, substituted isoquinolines,thiazole, and substituted thiazoles.
 6. The trans-platinum (U) complexof claim 1, wherein R is H or an alkyl group which may be substituted orunsubstituted.
 7. The trans-platinum (II) complex of claim 1, wherein Lor L′ is substituted with a substituent selected from the groupconsisting of Me, Br, Cl and F.
 8. The trans-platinum (II) complex ofclaim 1, wherein said trans-platinum (II) complex is selected from thegroup consisting of: trans-[Pt(OAc)₂(NH₃)(py)]trans-[Pt(OAc)₂(NH₃)(2Me-py)] trans-[Pt(OAc)₂(NH₃)(3Me-py)]trans-[Pt(OAc)₂(NH₃)(4Me-py)] trans-[Pt(OAc)₂(NH₃)(tz)]trans-[Pt(OAc)₂(NH₃)(quin)] trans-[Pt(OAc)₂(NH₃)(Iq)]trans-[Pt(OFm)₂(NH₃)(py)] trans-[Pt(OFm)₂(NH₃)(2Me-py)]trans-[Pt(OFm)₂(NH₃)(3Me-py)] trans-[Pt(OFm)₂(NH₃)(4Me-py)]trans-[Pt(OFm)₂(NH₃)(tz)] trans-[Pt(OFm)₂(NH₃)(quin)]trans-[Pt(OFm)₂(NH₃)(Iq)] trans-[Pt(lact)₂(NH₃)(py)]trans-[Pt(lact)₂(NH₃)(2Me-py)] trans-[Pt(lact)₂(NH₃)(3Me-py)]trans-[Pt(lact)₂(NH₃)(4Me-py)] trans-[Pt(lact)₂(NH₃)(tz)]trans-[Pt(lact)₂(NH₃)(quin)] trans-[Pt(lact)₂(NH₃)(Iq)]trans-[Pt(OAcOH)₂(NH₃)(py)] trans-[Pt(OAcOH)₂(NH₃)(2Me-py)]trans-[Pt(OAcOH)₂(NH₃)(3Me-py)] trans-[Pt(OAcOH)₂(NH₃)(4Me-py)]trans-[Pt(OAcOH)₂(NH₃)(tz)] trans-[Pt(OAcOH)₂(NH₃)(quin)]trans-[Pt(OAcOH)₂(NH₃)(Iq)] trans-[Pt(TFA)₂(NH₃)(py)]trans-[Pt(TFA)₂(NH₃)(2Me-py)] trans-[Pt(TFA)₂(NH₃)(3Me-py)]trans-[Pt(TFA)₂(NH₃)(4Me-py)] trans-[Pt(TFA)₂(NH₃)(py)]trans-[Pt(TFA)₂(NH₃)(quin)] trans-[Pt(TFA)₂(NH₃)(Iq)]trans-[Pt(OAc)₂(py)₂] trans-[Pt(OAc)₂(2Me-py)₂]trans-[Pt(OAc)₂(3Me-py)₂] trans-[Pt(OAc)₂(4Me-py)₂]trans-[Pt(OAc)₂(tz)₂] trans-[Pt(OFm)₂(py)₂] trans-[Pt(OFm)₂(2Me-py)₂]trans-[Pt(OFm)₂(3Me-py)₂] trans-[Pt(OFm)₂(4Me-py)₂]trans-[Pt(OFm)₂(tz)₂] trans-[Pt(lact)₂(py)₂] trans-[Pt(lact)₂(2Me-py)₂]trans-[Pt(lact)₂(3Me-py)₂] trans-[Pt(lact)₂(4Me-py)₂]trans-[Pt(lact)₂(tz)₂] trans-[Pt(OAcOH)₂(py)₂]trans-[Pt(OAcOH)₂(2Me-py)₂] trans-[Pt(OAcOH)₂(3Me-py)₂]trans-[Pt(OAcOH)₂(4Me-py)₂] trans-[Pt(OAcOH)₂(tz)₂]trans-[Pt(TFA)₂(py)₂] trans-[Pt(TFA)₂(2Me-py)₂]trans-[Pt(TFA)₂(3Me-py)₂] trans-[Pt(TFA)₂(4Me-py)₂] andtrans-[Pt(TFA)₂(tz)₂] wherein OAc=acetate CH₃COO⁻; OFm=formate HCOO⁻;Lact=lactate CH₃C.H(OH)COO⁻(.=R or S chirality); OAcOH=hydroxyacetateHOCH₂COO⁻; TFA=trifluoroacetate CF₃COO⁻; py=pyridine;2Me-py=2-methylpyridine; 3Me-py=3-methylpyridine;4Me-py=4-methylpyridine; tz=thiazole; Iq=isoquinoline; andquin=quinoline.